CN103018447B - Detect enzyme linked immunological kit and the method thereof of salbutamol - Google Patents
Detect enzyme linked immunological kit and the method thereof of salbutamol Download PDFInfo
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Abstract
The invention provides a kind of enzyme linked immunological kit and the detection method that detect salbutamol, enzyme linked immunological kit comprises: be coated with the ELISA Plate of coating antigen, salbutamol specific antibody, enzyme marker, salbutamol standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, Sample dilution.The invention provides a kind of salbutamol enzyme linked immunological kit detection method, mainly comprise: first carry out Sample pretreatment, then detect with kit, ultimate analysis testing result.Detection kit of the present invention can be applicable to the detection of the residual quantity of salbutamol in animal tissue's (muscle, liver), urine, feed sample, it is easy and simple to handle, quick, accurate, highly sensitive, low cost etc., is applicable to examination and the on-site supervision of great amount of samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit and the detection method that detect salbutamol in animal tissue's (muscle, liver), urine, feed.
Background technology
Salbutamol is a kind of potent selectivity beta-receptor stimulant substance, is usually used in treating the respiratory disease such as bronchial spasm of bronchial astehma, asthmatic bronchitis and emphysema patient clinically.Therefore medicine can improve lean meat percentage, reduces fat deposition and promotes growth of animal, by some livestock-raising enterprises as cultivation promoter illegal use.Its residual quantity can cause that human muscle trembles, palpitaition, neuroticism, headache, dizzy, Nausea and vomiting, have a fever, the symptom such as to tremble, harm is had to the healthy pole of consumer, " veterinary drug of food animal forbidding and other compound inventory " beta-stimulants such as clear stipulaties salbutamol that the Ministry of Agriculture of China issues is prohibited all purposes, forbids all food animals.
At present, for residue detection mainly contain the detection method such as high performance liquid chromatography, liquid chromatography ~ mass spectrometry analytic approach, vapor-phase chromatography, By Capillary Zone Electrophoresis, enzymoimmunoassay, instrument detection method has the high advantage of detection degree of accuracy, but its instrumentation degree is high, sample preparation is more complicated, testing cost is high, be not suitable for use in the detection of gross sample, immunological detection method is simple to operate, quick, sensitive, can detect most sample, be desirable rapid screening means simultaneously.
Summary of the invention
The object of this invention is to provide a kind of salbutamol enzyme linked immunological kit and application thereof.
A kind of salbutamol ELISA diagnostic kit, it comprises the ELISA Plate, salbutamol specific antibody, enzyme marker, salbutamol standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, the Sample dilution that are coated with coating antigen, described coating antigen is salbutamol drug antigenic, and described enzyme marker is enzyme labeling antiantibody.
Salbutamol enzyme linked immunological kit provided by the present invention, described salbutamol drug antigenic is the conjugate of salbutamol haptens and carrier protein, and described carrier protein is bovine serum albumin(BSA), mouse haemocyanin, rabbit serum proteins, thyroprotein, ovalbumin, hemocyanin or human serum albumins.
Described salbutamol specific antibody is salbutamol monoclonal antibody, obtains with salbutamol immunogen immune animal.Described salbutamol monoclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, and described salbutamol monoclonal antibody is preferably salbutamol mouse monoclonal antibody.
The marker enzyme of described enzyme marker is horseradish peroxidase, and enzyme labeling antiantibody adopts Over-voltage protection marker enzyme and the coupling of sheep anti mouse antiantibody to be obtained.
In order to carry out great amount of samples examination and on-site supervision more easily, described kit also comprises: salbutamol standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, Sample dilution.
Described standard solution is serial salbutamol standard solution, 1mL/ bottle, and concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L.Described substrate solution A liquid is urea peroxide solution, substrate solution B liquid is tetramethyl biphenyl amine aqueous solution, described stop buffer is the sulfuric acid solution of 1 ~ 2mol/L, described concentrated cleaning solution is the 0.15 ~ 0.2mol/LpH7.4 phosphate buffer containing 1% Tween-20, and the content of described Sample dilution to be pH be Tween-20 in the phosphate buffer of the 0.02mol/L of 7.4 is that the formulated dilution of 0.5% mixed solution dilutes.
The bag of wherein said ELISA Plate used by preparation process is buffered the carbonate buffer solution that liquid is the 0.05mol/L of pH9.6, and Block buffer is the calf serum solution of 10%.
The preparation of ELISA Plate of the present invention is mainly: be buffered liquid with bag and coating antigen is diluted to 0.2 ~ 0.3 μ g/ml, every hole adds 150 μ l, in 37 DEG C of environment, lucifuge is hatched 2h or 4 DEG C and is spent the night, incline liquid in hole, and cleansing solution washing 1 ~ 2 time, pats dry, 150 μ l confining liquids are added in every hole, 37 DEG C of lucifuges hatch 2h, and in hole of inclining, liquid pats dry, and preserve with the vacuum seal of aluminium film.
In the present invention, coating antigen and immunogenic building-up process are:
1. hapten synthesis (synthetic route is as Fig. 1)
(1) get 1-3g salbutamol, dissolve in 20-50mlDMF, then add the TEMPO of 10% molar equivalent and the tetrabutylammonium chloride of 10% molar equivalent, the NaHCO3 aqueous solution of 20-50ml0.5M, stirred at ambient temperature, after 30 minutes, adds the NCS of 1.1-1.5 molar equivalent, after room temperature reaction 2-5 hour, remove most of solvent, extraction into ethyl acetate, washing, dry, except desolventizing rear pillar chromatographic purifying, obtaining white solid, is salbutamol list oxide.
(2) 1-2g step (1) gained salbutamol oxide, dissolve in 20-30mlDMF, add the ethylenediamine of 2-5 molar equivalent, room temperature to 100 DEG C reaction 5-20 hour, except desolventizing, recrystallization in alcohol-water, obtains white solid, is the ethylenediamine condensation product of salbutamol list oxide.
2. immunogenic synthesis
(1) the water-soluble solution of salbutamol haptens 18mg 1.5ml is got.
(2) the GA8 μ l getting 50% adds in (1), stirred at ambient temperature reaction 18h.
(3) add in above-mentioned solution after getting the dilution of BSA100mg 5.5ml water.
(4) 24mgNaBH is added after reacting 24h
4reaction 2h.
(5) dialyse 48 hours with tri-distilled water, obtain immunogene.
3. the synthesis of coating antigen
(1) the water-soluble solution of salbutamol haptens 15mg 1.5ml is got.
(2) the GA8 μ l getting 50% adds in (1), stirred at ambient temperature reaction 18h.
(3) add in above-mentioned solution after getting the dilution of OVA30mg 4.5ml water.
(4) 24mgNaBH is added after reacting 24h
4reaction 2h.
(5) dialyse 48 hours with tri-distilled water, obtain coating antigen.
4. the preparation of monoclonal antibody
Animal immune: salbutamol haptens and carrier protein couplet thing immunity 8-10 Balb/c mouse in age in week.
Fusion of Cells and cloning: get the mice spleen cell after immunity, merge under the effect of fusion agent polyglycol (PEG) 4000 with SP2/0 myeloma cell, and screening obtains the hybridoma cell strain of energy stably excreting monoclonal antibody.
Cell cryopreservation and recovery: hybridoma cryopreserving liquid is made 1 × 10
9the cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
Present invention also offers a kind of method that salbutamol enzyme linked immunological kit detects salbutamolum residue in sample, this method is adopted to carry out qualitative to the salbutamol in animal tissue, feed, urine specimen or quantitatively detect, Sample pretreatment process is simple, sample in enormous quantities can be detected fast simultaneously, kit has very high degree of accuracy and sensitivity, lower operative technique requires and of short duration detection time, and detect the features such as sample size is large, key step comprises:
1) sample to be tested is carried out pre-treatment, obtain sample to be tested solution;
2) sample to be tested solution is detected with enzyme linked immunological kit;
3) testing result is analyzed.
Accompanying drawing explanation
Fig. 1: salbutamol hapten synthesis figure
Fig. 2: salbutamol typical curve
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be understood that these embodiments are only for illustration of the present invention, and be not used for limiting the scope of the invention.
Embodiment one: the preparation of antigen, antibody and enzyme marker
1. the haptenic preparation of salbutamol
(1) get 1-3g salbutamol, dissolve in 20-50mlDMF, then add the TEMPO of 10% molar equivalent and the tetrabutylammonium chloride of 10% molar equivalent, the NaHCO3 aqueous solution of 20-50ml0.5M, stirred at ambient temperature, after 30 minutes, adds the NCS of 1.1-1.5 molar equivalent, after room temperature reaction 2-5 hour, remove most of solvent, extraction into ethyl acetate, washing, dry, except desolventizing rear pillar chromatographic purifying, obtaining white solid, is salbutamol list oxide.
(2) 1-2g step (1) gained salbutamol oxide, dissolve in 20-30mlDMF, add the ethylenediamine of 2-5 molar equivalent, room temperature to 100 DEG C reaction 5-20 hour, except desolventizing, recrystallization in alcohol-water, obtains white solid, is the ethylenediamine condensation product of salbutamol list oxide.
2. immunogenic preparation
(1) the water-soluble solution of salbutamol haptens 18mg 1.5ml is got.
(2) the GA8 μ l getting 50% adds in (1), stirred at ambient temperature reaction 18h.
(3) add in above-mentioned solution after getting the dilution of BSA100mg 5.5ml water.
(4) 24mgNaBH is added after reacting 24h
4reaction 2h.
(5) dialyse 48 hours with tri-distilled water, obtain immunogene.
3. the preparation of coating antigen
(1) the water-soluble solution of salbutamol haptens 15mg 1.5ml is got.
(2) the GA8 μ l getting 50% adds in (1), stirred at ambient temperature reaction 18h.
(3) add in above-mentioned solution after getting the dilution of OVA30mg 4.5ml water.
(4) 24mgNaBH is added after reacting 24h
4reaction 2h.
(5) dialyse 48 hours with tri-distilled water, obtain coating antigen.
4. the preparation of ELISA Plate
The bag of described ELISA Plate used by preparation process is buffered the carbonate buffer solution that liquid is the 0.05mol/L of pH9.6, and Block buffer is the calf serum solution of 10%.
The preparation of ELISA Plate of the present invention is mainly: be buffered liquid with bag and coating antigen is diluted to 0.2 ~ 0.3 μ g/ml, every hole adds 150 μ l, in 37 DEG C of environment, lucifuge is hatched 2h or 4 DEG C and is spent the night, incline liquid in hole, and cleansing solution washing 1 ~ 2 time, pats dry, 150 μ l confining liquids are added in every hole, 37 DEG C of lucifuges hatch 2h, and in hole of inclining, liquid pats dry, and preserve with the vacuum seal of aluminium film.
5. the preparation of sheep anti mouse antiantibody
Take sheep as immune animal, with mouse source antibody for immunogen immune pathogen-free domestic sheep, obtain sheep anti mouse antiantibody.
6. the preparation of enzyme labeling sheep anti mouse antiantibody (enzyme labeling antiantibody)
Adopt the Over-voltage protection after improveing to carry out coupling horseradish peroxidase and antiantibody, eliminate amino closed process, because it is seldom actual to produce self the amino amino connected; Horseradish peroxidase: the volumetric molar concentration ratio of antiantibody is 2: 1, the method after improvement is easier than traditional method, reduces the loss of the activity of enzyme.
7. the preparation method of monoclonal antibody
Animal immune: salbutamol haptens and carrier protein couplet thing immunity 8-10 Balb/c mouse in age in week.
Fusion of Cells and cloning: get the mice spleen cell after immunity, merge under the effect of fusion agent polyglycol (PEG) 4000 with SP2/0 myeloma cell, and screening obtains the hybridoma cell strain of energy stably excreting monoclonal antibody.
Obtain the strain of salbutamol monoclonal hybridoma, the generation salbutamol specific antibody that this hybridoma cell strain can be endless through screening, this antibody specificity is for salbutamol, and sensitivity reaches 0.1 μ g/L.
Cell cryopreservation and recovery: hybridoma cryopreserving liquid is made 1 × 10
9the cell suspension of individual/ml, preserves for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, put into 37 DEG C of water-bath middling speeds immediately and melt, after centrifugal segregation cryopreserving liquid, move into and cultivate culture in glassware.
Embodiment two: the establishment of each component of salbutamol enzyme linked immunological kit
Set up salbutamol enzyme linked immunological kit, comprise following each component:
(1) ELISA Plate of coating antigen is coated with.
(2) enzyme labeling antiantibody: horseradish peroxidase-sheep anti mouse antiantibody.
(3) salbutamol monoclonal antibody working fluid.
(4) standard solution: adopt gradient dilution method preparation standard solution, obtain serial standards 6 bottles, concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, and high standard product 100 μ g/L, 1mL/ bottle.
(5) substrate nitrite ion A liquid is urea peroxide solution, and substrate nitrite ion B liquid is tetramethyl biphenyl amine aqueous solution.
(6) stop buffer is the sulfuric acid solution of 1 ~ 2mol/L.
(7) concentrated cleaning solution is the 0.15 ~ 0.2mol/LpH7.4 phosphate buffer containing 1% Tween-20.
(8) content of Sample dilution to be pH be Tween-20 in the phosphate buffer of the 0.02mol/L of 7.4 is that the formulated dilution of 0.5% mixed solution dilutes.
Embodiment three: detect salbutamol residual in sample
1. the pre-treatment of sample
(1) tissue, liver pre-treating method
Take the equal pledge of 2.0+0.05g, add 2ml3% trichloroacetic acid (take 15g trichloroacetic acid (solid), add the mixing of 500ml deionized water dissolving), whirling motion 5min, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min.Get supernatant 500 μ l, add 500 μ l Sample dilution, fully mixing (pH value should between 7-9).Get 50 μ l for analyzing.
(2) pre-treating method of urine sample
Get the limpid urine sample of 50ml directly to measure (as urine sample muddiness must by filtering or more than 3000g, 15 DEG C of centrifugal 10min are until limpid), freezen protective answered by the sample that wouldn't use.
(3) pre-treating method of feed
Take 1.0g feed sample, add 10ml methyl alcohol, whirling motion 5min, more than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min; Pipette 1ml upper organic phase (being equivalent to 0.1g sample) in 10ml glass tube, flow down in 50 ~ 60 DEG C of nitrogen and dry up; Add 1ml normal hexane, whirling motion 30s, then add 1ml3% trichloroacetic acid, whirling motion 1min; More than 3000g, room temperature (20-25 DEG C/68-77 °F) centrifugal 5min, removing upper organic phase, takes off layer liquid 150 μ l, adds 450 μ l Sample dilution, fully mixing (pH value should between 7-9).Get 50 μ l for analyzing.
2. detection method
(1) prepare: by all reagent and need taking out from cold storage environment with lath before use, make temperature recovery to room temperature (20-25 DEG C), after use, immediately all reagent is put back to 2-8 DEG C.
(2) standard items/sample is added: add standard items/sample 50 μ l in the micropore of correspondence.
(3) mixing of antibody working fluid and enzyme labeling antiantibody concentrate: antibody working fluid and enzyme labeling antiantibody concentrate are mixed by 10: 1 volume ratios and mixes.(note: this mixed liquor can not be preserved, carries out application of sample after mixing at once)
(4) add the mixed liquor of antibody working fluid and enzyme labeling antiantibody concentrate: the mixed liquor 50 μ l/ hole adding antibody working fluid and enzyme labeling antiantibody concentrate, mixing of vibrating gently, react 30min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
(5) plate is washed: carefully open cover plate film, liquid in hole is dried, with wash operating solution (20 × concentrated cleaning solution being diluted by 1: 19 volume ratio with deionized water) 250 μ l/ hole, abundant washing 4-5 time, every minor tick 10s, pats dry (bubble be not eliminated after patting dry can be poked with original rifle head) with thieving paper.
(6) develop the color: add substrate solution A liquid 50 μ l/ hole, then add substrate solution B liquid 50 μ l/ hole, mixing of vibrating gently, with the 15min that develops the color in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, add stop buffer 50 μ l/ hole, mixing of vibrating gently.
(7) measure: microplate reader measures the absorbance in every hole, setting microplate reader is in 450nm place (suggestion dual wavelength 450/630nm detects, and runs through data in 5min).
3. Analysis of test results
The percentage absorptance of standard items or sample equals the absorbance of mean value (diplopore) divided by first standard (0 standard) of the absorbance of standard items or sample, then is multiplied by 100%, namely obtains percentage absorptance.With salbutamol standard items percentage absorptance for ordinate, with the logarithm of salbutamol standard concentration (μ g/L) for horizontal ordinate, drawing standard curve map (as Fig. 2).The percentage absorptance of sample substituted in typical curve, read the concentration corresponding to sample from typical curve, the extension rate being multiplied by its correspondence is salbutamol actual concentrations in sample.
Embodiment four: the sensitivity of salbutamol kit, specificity, preci-sion and accuracy, storage life experiment
1. kit sensitivity determination
Conventionally measure kit sensitivity test, kit standard curve minimum point is 0.1 μ g/L, and the scope of typical curve is 0.1 μ g/L ~ 8.1 μ g/L, and salbutamol detects and is limited to 0.5 μ g/kg in the tissue, detect in urine and be limited to 0.3 μ g/L, detect in feed and be limited to 5 μ g/kg.
2. kit accuracy and precision
Accuracy refers to the matching degree between measured value and true value, and the accuracy of kit is commonly used the recovery and represented; Precision is that reaction assay method repeatedly measures the repetition degree of acquired results to a certain specific sample, and the conventional coefficient of variation represents.Respectively to blank pork, pork liver, pig urine, to add salbutamol to final concentration in feed be 2 μ g/kg, and 2 μ g/L, 2 μ g/L, 10 μ g/kg, repeats 5 times, gets the kit calculating coefficient of variation of three batches respectively, the results are shown in following table.
The accuracy of table 1 kit and precision measure
Result shows, in pork, be added into final concentration is 2 μ g/kg, recovery scope is 77.5% ~ 102.5%, in pork liver, be added into final concentration is 2 μ g/kg, recovery scope is 73.0% ~ 101.0%, in pig urine, be added into final concentration is 2 μ g/kg, recovery scope is 61.5% ~ 80.0%, in feed, be added into final concentration is 10 μ g/kg, recovery scope is 75.5% ~ 100.5%, in batch, interassay coefficient of variation is less than 20%, meet " Ministry of Agriculture's file " agriculture doctor [2005] No. 17 annex 2 kits to put on record with reference to the regulation of the 4th preci-sion and accuracy in judgment criteria.
3. cross reacting rate test
Select salbutamol as follows, clenbuterol, Ractopamine 3 kinds of medicines conventionally to measure cross reacting rate respectively, result is as shown in table 2.
The specificity of table 2 kit
4. storage life experiment
Kit preservation condition is 2-8 DEG C, measures through 12 months, and the maximum absorbance value of kit, IC50 value, salbutamol add practical measurement value all within normal range.Do accelerated deterioration and refrigeration test, kit to be placed in 37 DEG C ,-20 DEG C 6 days, measurement result also shows that the indices of kit is normal simultaneously.Obtain salbutamol kit from above result to preserve 12 months at 2-8 DEG C.
Claims (9)
1. a salbutamol ELISA diagnostic kit, it comprises the ELISA Plate, salbutamol specific antibody, enzyme marker, salbutamol standard solution, substrate nitrite ion, stop buffer, concentrated cleaning solution, the Sample dilution that are coated with coating antigen, described enzyme marker is enzyme labeling antiantibody, described coating antigen is salbutamol drug antigenic, salbutamol drug antigenic is the conjugate of salbutamol haptens and carrier protein, and described salbutamol is haptenic preparation method mainly comprise the steps:
(1) get 1-3g salbutamol, dissolve in 20-50mlDMF, then add the TEMPO of 10% molar equivalent and the tetrabutylammonium chloride of 10% molar equivalent, the NaHCO of 20-50ml0.5M
3aqueous solution, stirred at ambient temperature, after 30 minutes, adds the NCS of 1.1-1.5 molar equivalent, after room temperature reaction 2-5 hour, remove most of solvent, extraction into ethyl acetate, washing, dry, except desolventizing rear pillar chromatographic purifying, obtaining white solid, is salbutamol list oxide;
(2) 1-2g step (1) gained salbutamol list oxide, dissolve in 20-30mlDMF, add the ethylenediamine of 2-5 molar equivalent, room temperature to 100 DEG C reaction 5-20 hour, except desolventizing, recrystallization in alcohol-water, obtains white solid, is the ethylenediamine condensation product of salbutamol list oxide.
2. kit as claimed in claim 1, is characterized in that: described carrier protein is bovine serum albumin(BSA), mouse haemocyanin, rabbit serum proteins, thyroprotein, ovalbumin, hemocyanin or human serum albumins.
3. kit as claimed in claim 1, is characterized in that: described salbutamol specific antibody is salbutamol anti-drug monoclonal antibody.
4. kit as claimed in claim 1, is characterized in that: described antiantibody is sheep anti mouse antiantibody.
5. kit as claimed in claim 1, is characterized in that the marker enzyme of described enzyme marker is horseradish peroxidase; Enzyme labeling antiantibody adopts Over-voltage protection marker enzyme and antiantibody coupling to be obtained.
6. kit as claimed in claim 1, is characterized in that: described substrate nitrite ion A liquid is urea peroxide solution, and substrate nitrite ion B liquid is tetramethyl biphenyl amine aqueous solution, and described stop buffer is the sulfuric acid solution of 1 ~ 2mol/L.
7. kit as claimed in claim 1, it is characterized in that: described concentrated cleaning solution is the 0.15 ~ 0.2mol/LpH7.4 phosphate buffer containing 1% Tween-20, described Sample dilution is be the phosphate buffer of 7.4 containing the 0.02mol/LpH of 0.5% Tween-20.
8. kit as claimed in claim 1, is characterized in that: described standard solution concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L.
9. the salbutamol ELISA diagnostic kit as described in any one of claim 1-8 detects the method for salbutamolum residue in sample, and key step comprises:
1) sample to be tested is carried out pre-treatment, obtain sample to be tested solution;
2) sample to be tested solution is detected with the salbutamol ELISA diagnostic kit described in any one of claim 1-8;
3) testing result is analyzed.
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| CN103508910B (en) * | 2012-06-19 | 2016-04-20 | 北京勤邦生物技术有限公司 | Beta-stimulants class haptens and its preparation method and application |
| CN105301244B (en) * | 2014-06-03 | 2017-07-21 | 北京勤邦生物技术有限公司 | Detect enzyme linked immunological kit and its application of acid orange |
| CN107561269A (en) * | 2017-08-23 | 2018-01-09 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of salbutamol |
| CN110004117B (en) * | 2019-04-11 | 2021-03-09 | 中抗生物医药(杭州)有限公司 | Hybridoma cell strain secreting salbutamol monoclonal antibody, monoclonal antibody and application |
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| CN101241133A (en) * | 2008-01-18 | 2008-08-13 | 华南农业大学 | ELISA kit for detecting salbutamolum residue and method of use thereof |
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| CN101592658A (en) * | 2009-06-26 | 2009-12-02 | 青岛康地恩药业有限公司 | A kind of salbutamol ELISA diagnostic kit and application thereof |
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